Polyclonal antibodies (Abs) against zebrafish TBK1 were generated according to our previous method (18 (link)), which can recognize TBK1 normal form, TBK1_tv1 and TBK1_tv3 isoforms, but not for TBK1_tv2 isoform. Anti-FLAG and anti-HA mouse Abs, dimethylsulfoxide (DMSO), and FLAG Immunoprecipitation Kit were purchased from Sigma-Aldrich. The anti-pTurboGFP antibody was purchased from Everogen. The anti-GAPDH mouse antibody was purchased from Proteintech. Poly(I:C) was purchased from InvivoGen. The MG132 and NH4Cl were purchased from Selleck. Lipofectamine 2000 and protease inhibitor cocktail were purchased from Thermo Fisher Scientific.
Mouse anti gapdh antibody
Manufactured by Proteintech
Sourced in United States, China
The Mouse anti-GAPDH antibody is an immunoaffinity-purified antibody that recognizes the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein. GAPDH is a highly conserved enzyme involved in glycolysis. This antibody can be used to detect GAPDH in various applications, such as Western blotting, immunoprecipitation, and immunocytochemistry.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (6 mentions)
Ripa lysis buffer, by Beyotime (3 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Ecl reagent, by Thermo Fisher Scientific (2 mentions)
Ripa buffer, by Beyotime (2 mentions)
Bca protein assay kit, by Beyotime (2 mentions)
Goat anti rabbit igg, by LI COR (2 mentions)
Goat anti mouse igg, by LI COR (2 mentions)
Rabbit anti e cadherin antibody, by Proteintech (2 mentions)
Nh4cl, by Selleck Chemicals (1 mentions)
Poly 1 c, by InvivoGen (1 mentions)
Flag immunoprecipitation kit, by Merck Group (1 mentions)
Mg132, by Selleck Chemicals (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Proteinase inhibitor cocktail, by Roche (1 mentions)
Protease inhibitor cocktail, by Selleck Chemicals (1 mentions)
Mouse anti n cadherin, by Cell Signaling Technology (1 mentions)
Rabbit anti zo 1, by Thermo Fisher Scientific (1 mentions)
Western blot chemiluminescence hrp substrate, by Takara Bio (1 mentions)
19 protocols using mouse anti gapdh antibody
1
Polyclonal Antibodies for Zebrafish TBK1
2
Western Blot Analysis of Protein Samples
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Total protein from tissues or cultured cells were harvested by RIPA buffer (P0013B, Beyotime, USA) with the proteinase inhibitor cocktail (4693132001, Roche, Switzerland). Denatured protein samples in equal quantities were loaded on SDS-polyacrylamide gels, and transferred onto polyvinylidene difluoride membranes. The membrane was blocked with 5% defatted milk and then incubated with corresponding primary antibodies, followed by the horseradish peroxidase conjugated secondary antibody. Finally, proteins were visualized by ECL reagents (WP20005, Thermo Fisher Scientific, USA). Anti-MNK1 rabbit antibody (1:1,000 dilution, #2195, Cell Signaling Technology, USA) and anti-GAPDH mouse antibody (1:1,000 dilution, ProteinTech Group Inc, USA) were used in this assay.
3
Analyzing ARPE-19 Cell Junctions via Western Blotting
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Western blotting was used to further examine the expression of tight junction and adherens junction proteins in ARPE-19 cells. ARPE-19 cells cultured under conditions identical to those described for the permeability assay were washed with precooled PBS and then lysed for 30 min at 4°C with lysis buffer (50 mM Tris/HCl, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 0.25% Na-deoxycholate, 1 mM EDTA, and protease inhibitor cocktail [B14001, Bimake, Houston, TX, USA]). The total proteins were harvested by centrifugation at 13,000 rpm and 4°C for 20 min, and the protein concentration in each sample was measured using G250. Next, 30–50 μg of total protein per sample was loaded into a well of an 8% polyacrylamide gel, separated by SDS-PAGE, and transferred to polyvinylidene fluoride (PVDF) membranes (Merck KGaA, Darmstadt, Germany). The membranes were blocked with 5% nonfat milk in 20 mM Tris (pH 7.4) with 137 mM NaCl and 0.05% Tween 20 for 1 h at RT. The membranes were then probed with a rabbit anti-ZO-1 (dilution, 1:1000; Invitrogen), mouse anti-N-cadherin (1:1000; Cell Signaling Technology), or mouse anti-GAPDH antibody (1:1000; Proteintech, Rosemont, IL, USA) overnight at 4°C. After washing, the membranes were incubated with a horseradish peroxidase-conjugated secondary antibody for 1 h at RT. All the PVDF membranes were analyzed using chemiluminescence (Tanon, Shanghai, China).
4
Western Blot Analysis of Protein Expression
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Cells were harvested at 36 h post-transfection (hpt). The samples were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with 5% skim milk for 2 h at 37 °C, incubated with a mouse anti-Flag antibody (MBL, Japan, 1:5000), mouse anti-HA antibody (MBL, Japan, 1:4000) or mouse anti-GAPDH antibody (Proteintech, Beijing, 1:20 000) overnight at 4 °C, and then probed with an HRP-conjugated secondary antibody (Bio-Rad, CA, USA) for 1 h at 37 °C. Proteins were detected with Western Blot Chemiluminescence HRP Substrate (Takara, Dalian, China) according to the manufacturer’s instructions [45 (link)].
5
Western Blot Analysis of ROR2 and Akt
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Subconfluent cells were washed twice with PBS, and then lysed with ice-cold RIPA lysis buffer (50 mmol/L Tris, 150 mmol/L NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 1 mmol/L sodium orthovanadate, 1 mmol/L sodium fluoride, 1 mmol/L EDTA, 1 mmol/L PMSF, and 1% cocktail of protease inhibitors) (pH7.4). The lysates were then clarified by centrifugating at 12,000g for 20 min at 4 °C. The protein extracts were separated by SDS-PAGE. The immunoblotting procedure was performed as described [16 (link)] and the following antibodies were used: rabbit anti-ROR2 antibody, mouse anti-GAPDH antibody (Proteintech, Wuhan, China), rabbit anti-Akt antibody, rabbit anti-phospho-Akt (p-Ser473) antibody (Cell Signaling Technology, Danvers, MA). Protein bands were detected by incubating with horseradish peroxidase-conjugated antibodies (Santa Cruz Biotechnology) and visualized with ECL reagent (Thermo Scientific, Rockford, IL). The gray values were taken by Tanon imaging analysis system (Tanon, Shanghai, China).
6
Quantitative Protein Expression Analysis
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Total proteins were lysed by RIPA buffer (Beyotime, China). After centrifugation (4°C, 12,000 g for five minutes), protein concentrations were measured by Bio-Rad Protein Assay Kit (Bio-Rad Laboratories, CA, USA). SDS-PAGE was conducted to separate equal amounts of proteins, which were blotted onto PVDF membrane (Millipore, USA). After blocked with milk, the membranes were incubated overnight at 4°C with primary antibodies. Primary antibodies were as follows: rabbit anti-p-c-Kit (Tyr719) antibody, rabbit anti-c-Kit antibody (CST, USA); rabbit anti-α-SMA antibody, rabbit anti-p-PDGFRα (Tyr720) antibody, rabbit anti-PDGFRα antibody (Abcam, USA); and mouse anti-GAPDH antibody (Proteintech, USA). Afterwards, membranes were incubated with the second antibody (Invitrogen, USA) at 25 °C for one hour. Finally, the blots were detected with Kodak film developer (Fujifilm, Japan). The protein blots were analyzed by the ImageJ software.
7
Immunoblot Analysis of Cell Signaling
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The antibodies used were mouse anti-GAPDH antibody (Proteintech, 60004-1, 1:5,000, China), mouse anti-Ki-67 antibody (BD Biosciences, 558616, 1:100, USA), and mouse anti-AURKA antibody (Abcam, ab13824, 1:4,000, USA). USP8 inhibitor DUB-IN-1 was purchased from MedChemExpress (MCE, HY-50736, USA).
8
Western Blot Analysis of MMP2 Protein
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Total protein lysates were extracted using RIPA lysis buffer (Beyotime) supplemented with protease inhibitors (Roche, Penzberg, Germany), and the concentrations of the samples were determined using a BCA Protein Assay Kit (Beyotime). Equal amounts of total proteins were separated by SDS-PAGE (Transgen, Beijing, China), transferred to the PVDF membrane (Millipore, Billerica, MA, USA) and probed with a rabbit anti-MMP2 polyclonal antibody (1:2000, Proteintech, Wuhan, China) and mouse anti-GAPDH antibody (1:10,000, Proteintech). After overnight incubation with HRP-conjugated secondary antibodies (1:10,000, Proteintech), protein bands were visualized using Immobilon Western Chemiluminescent HRP Substrate (Millipore) on a Tanon-5200 chemiluminescence detection system (Tanon, Shanghai, China). The bands were analyzed by using Image J software (NIH, Bethesda, MD, USA).
9
Recombinant IL-25 and Angiogenesis Pathway
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Recombinant mouse IL-25 (rmIL-25, Cat#: 587306) was brought from Biolegend, recombinant human IL-25 (rhIL-25, Cat#: 8134-IL) was purchased from R&D systems and prepared following instructions. Rabbit anti-CD31 antibody (Cat#: GB11063-2) was obtained from Servicebio. Goat anti-IL-17RB antibody (Cat#: AF1040) was obtained from R&D systems. Rabbit anti-VEGF antibody (Cat#: ab52917) and rabbit anti-β-catenin antibody (Cat#: ab32572) was from Abcam. Mouse anti-GAPDH antibody (Cat#: 60004-1-Ig) and rabbit anti-IL-17RB antibody (Cat#: 20673-1-AP) were obtained from Proteintech. Rabbit anti-phospho-Akt antibody (Cat#: 4060T), rabbit anti-Akt antibody (Cat#: 4091T), rabbit anti-phospho-ERK 1/2 antibody (Cat#: 4370S) and rabbit anti-ERK 1/2 antibody (Cat#: 4695T) were purchased from Cell Signaling Technology (CST). Glucose and streptozotocin (STZ, Cat#: S0130) were purchased from Sigma-Aldrich; and pLenti-CMV-IL-25-GFP-puro lentiviral plasmid (Lenti-IL-25-GFP, Cat#: PPL02165-4a) was obtained from Public Protein/Plasmid Library (PPL). The lentiviral packaging plasmid pMD2.G (Cat#: 12259) and psPAX2 (Cat#: 12260) were obtained from AddGene. Basement membrane matrix (Cat#: 354234) was obtained from Corning.
10
Protein Expression Analysis via Western Blot
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RIPA Lysis Buffer (CWbio, China) containing protease inhibitors was used to collect samples (Roche, USA). Twenty grams of proteins were separated using 4%–20% gradient SDS‐PAGE gels (GenScript, USA) and transferred to PVDF membranes (Millipore, Germany). The membranes were blocked for 1 h at room temperature with 1% BSA before being incubated overnight at 4°C with primary antibodies: mouse anti‐RUNX2 antibody (Abcam, UK) at 1:1000, rabbit anti‐osteopontin (OPN) antibody (Affinity, USA) at 1:1000 and mouse anti‐GAPDH antibody (Proteintech, USA) at 1:10,000. Then, the membranes were washed with TBS and with Tween‐20 (TBST) three times, and incubated for 1 h with a secondary antibody: anti‐mouse IgG antibody (HRD) (Sigma‐Aldrich, USA) and anti‐rabbit IgG antibody (HRD) (Sigma‐Aldrich, USA) at room temperature. Images were obtained using the ChemiDoc XRS + imaging system (Bio‐Rad, USA).
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